Novel complexes comprising collagen peptides and curcuminoids and compositions thereof

ABSTRACT

Formulations comprising complexes of peptides with hydrophobic bioactive molecules are described herein. The invention more specifically relates to collagen peptide and curcuminoid complexes and compositions comprising the complexes. The compositions and formulations comprising the protein hydrolysate peptide-hydrophobic bioactive molecule complexes are water-soluble, stable at physiological and acidic pH, synergistically enhance the systemic bioavailability of the biomolecules and peptides, and are capable of delivering a high therapeutically effective amount of the bioactive molecules via oral route. The compositions and formulations comprising the collagen peptide-curcuminoid complexes provide significantly high levels of bioavailable curcuminoids that are water soluble and stable at physiological and acidic pH along with significant anti-inflammatory effects offered by collagen peptides. The invention further provides a process for preparing the peptide-bioactive molecule complexes, compositions and oral formulations comprising the collagen peptide-curcuminoid complex.

RELATED APPLICATIONS

This application claims the benefit of the filing date of IndianProvisional Patent Application No. IN201941003319, filed Jan. 28, 2019,the disclosure of which is incorporated, in its entirety, by thisreference.

TECHNICAL FIELD

The present invention relates to complexes comprising small hydrophobicbioactive molecules and protein hydrolysate peptides, and water-solubleformulations comprising them. More specifically, the invention relatesto complexes comprising collagen peptides and curcuminoids andformulations comprising the complexes. The formulation described hereincomprising the collagen peptide-curcuminoid complexes are water-soluble,stable at physiological and acidic pH, and enhances the systemicbioavailability of curcuminoids with enhanced elimination half-life,thus delivering therapeutically effective amount of curcuminoids viaoral route. The invention thus provides a novel route for thesimultaneous oral delivery of therapeutically relevant dosage of bothcollagen peptides and curcuminoids. The invention further provides theprocess for preparing the collagen peptide-curcuminoid complex.

BACKGROUND

Oral delivery of bioactive molecules is the preferred route ofadministration by consumers and manufacturers for their ease andconvenience contributing to higher adherence, lower production costs andgreatest versatility with respect to optimization of drug delivery overother forms. However, oral delivery has been limited to a few moleculesas various difficulties with respect to the physical and chemicalproperties of bioactive molecules and the physiology of thegastrointestinal (GI) tract. The main consequence of the poor absorbanceand degradation of bioactive molecules administered orally is lowbioavailability. Increasing dose to overcome the poor bioavailabilityproblem increases concerns about both potential adverse effects,formulation difficulties and excessive costs. Another challenge todevelop formulations of biologically active molecules is their stabilityin formulations. Effective solutions for overcoming issues of bioactivemolecules such as hydrophobicity, instability, rapid biotransformationto inactive metabolites have yet to be identified, so thattherapeutically effective concentrations can be delivered withoutincreasing dosages. In the case of nutraceuticals and functional food,food-grade delivery forms or delivery technologies employing foodcomponents or molecules from food is a great challenge.

Proteins are essential components of life and are widely distributed inplants and animals. They have important physiological role. Isolatedproteins have been characterised to be macromolecules composed of aminoacids. Once consumed, they are getting hydrolysed to peptides and aminoacids having definite nutritional properties. Several food proteinhydrolysates composed of peptides and amino acids are available as foodingredients. Collagen peptides are one such group of collagen proteinhydrolysates that are shown to be having functional benefits for skin,bone, joints, and gastrointestinal tract. Collagen peptides are reportedto improve the skin health (reduces wrinkles, increases hydration,increases the skin radiance), bone health (bone density and cartilageformation), joint health (reduces osteoarthritis pain and increasesjoint flexibility) and gastro protective. However, its anti-inflammatoryeffect has been shown to be low and very often require high dosage (3 to10 g/day) and longer duration of supplementation (6 months to one year).So there exist a practice of co-administration of anti-inflammatoryphytonutrients like curcuminoids, Boswellia extract, ginger extract etc.along with collagen peptides with a view to modulate the efficacy.

Considering the requirement of relatively high dosage of proteinhydrolysates, the very often used delivery format is food, especially inthe form of sachets (ready-to-drink powder). Since the bioactivemolecules are hydrophobic, water insoluble and having strong colour andtaste, their incorporation into peptides is very difficult. One of thepractices is to make physical mixtures using a large excess of syntheticemulsifier like polysorbate for supplementation. Such formulationssuffer from serious draw backs of poor bioavailability of thephytonutrients, undesirable colour and taste, staining tong,hygroscopicity, and requirement of special packaging.

Curcuminoids are natural polyphenolic compounds derived from turmeric(Curcuma longa L). The mixture of curcumin, demethoxycurcumin (DMC) andbisdemthoxycurcumin (BDMC) isolated from turmeric rhizomes are known as‘curcuminoids’, and are often referred to as ‘curcumin’. Curcuminoidshave been shown to be modulators of multiple intercellular signallingpathways linked to inflammation, to proliferation, growth, invasion,drug sensitivity, angiogenesis and cancer cell metastasis in many cellbased assays.

Although curcuminoids has shown significant efficacy in cell culturestudies, it has shown limited efficacy in clinical studies whenadministered in conventional oral formulations, with low nanomolarlevels of the parent compound and its glucuronide and sulphateconjugates found in the peripheral or portal circulation. It has beenreported that serum levels in humans after an oral dose of 3 g curcuminalone were either undetectable or very low. Thus, the serum curcuminoidslevel is insufficient to provoke the desired beneficial effect of thiscompound, and cannot be achieved by the mere consumption of turmeric orcurcuminoids.

Curcumin is highly hydrophobic and usually is not present in dissolvedform when delivered as a component of commonly available nutraceuticals.Effective concentration for curcumin to exert any of the cellulareffects has been demonstrated to be in the range of at least 100-2,000nanomolar (0.1-2 micromolar) levels in vitro, but currently availablecurcumin supplements lead to negligible, low nanomolar blood levels. Thepoor oral bioavailability may result from its poor solubility,gastrointestinal instability, poor pharmacokinetic profile, or acombination of these factors. In particular, characteristics of curcuminsuch as chemical instability at neutral and slightly alkaline pH, itshydrophobic nature, and typically its insolubility at acidic pH whendelivered as a dry powder in existing supplements. Most of the curcuminis never absorbed and is excreted. The susceptibility to autoxidation,its rapid biotransformation to conjugated glucuronides/sulfates(conjugative metabolism) by reaction of curcumin's phenolic hydroxylgroups and high “first pass” clearance leads to its poor permeation fromthe intestinal lumen to the portal blood.

These disadvantages have posed a major impediment in the development ofvarious therapeutic and prophylactic applications of curcuminoids inspite of its pleiotropic pharmacological properties. To improve thebioavailability, sophisticated drug delivery systems are still beingresearched.

WO2015025263A1 describes a curcumin composition for increasing thebioavailability of curcumin mainly due to the presence of volatile oilof turmeric. The curcumin mixture comprises curcumin dry crystals,volatile oil, fixed oil whereas water extract comprises solubleproteins, dietary fibers and carbohydrates extracted from turmeric,along with a natural emulsifier isolated from Quillaja saponaria andlecithin. The curcumin composition showed bioequivalence with 500 mgcurcumin capsules.

US20100179103 A1 describes a method of increasing the delivery ofcurcumin by complexing it with cyclodextrins. The combination ofcyclodextrins and curcumin in pre-clinical inflammation modelsdemonstrated efficacy superior to both the positive control andcurcumin. The curcumin are delivered by cyclodextrins which are cyclicoligomers of glucose, by forming inclusion complexes with the insolublecurcumin molecule which fit into the lipophile-seeking cavities of thecyclodextrin molecule.

WO2010/010431 describes a liquid and semisolid self-emulsifying curcuminformulations based on a lipid carrier system of PEG fatty acid esterswhich showed improved bioavailability compared to an aqueous suspensionof curcumin after oral administration to rats.

WO 2010/013224 describe curcumin nanoparticles and curcumin bound tochitosan nanoparticles to provide improved oral bioavailability ofcurcumin in mice compared to curcumin orally administered in olive oil.

Researchers Gomez-Estaca et al., (Food Hydrocolloids, 2017, Volume 70:313-320) have attempted encapsulation of curcumin in gelatin throughelectrohydrodynamic atomization. Water solubility of curcumin was foundto increase by 38.6 folds. The antioxidant and antimicrobial propertiesof the encapsulated curcumin was also found to be improved. However,there is an absence of any information on the bioavailability ofcurcuminoids, the efficacy of curcumin-gelatin formulation onanti-inflammatory effect or any other pharmacological effect or anydosage information for the oral delivery of therapeutically relevantdoses of curcuminoids and gelatin. Moreover, gelatin is a very highmolecular weight protein, which forms gels and thick solutions in waterwhich provides challenges to preparation of oral formulations.

Lately, self-emulsifying drug delivery system (SEDDS) have been shown toimprove the solubility and bioavailability of curcuminoids. Theameliorative effect of curcuminoid SEDDS are reported to be markedlybetter than that of suspension of curcuminoids in rat models for chronicheart failure (Jiang, Yunbin et al., Saudi Pharmaceutical Journal: SPJ26.4 (2018): 528-534. PMC. Web. 5 Jul. 2018). However, SEDDS facesdisadvantages due to presence of high amount of surfactant,contradiction in correlation of in vitro model to in vivo studies, lackof human volunteer study and effect of conversion of SEDDS to finaladministrable dosage form on pharmacokinetic behaviour of the drug.

Researchers Comblain, Fanny et al. (Ed. Christos Chadjichristos. PLoSONE 10.3 (2015): e0121654. PMC. Web. 6 Jul. 2018) studied the in vitroeffects of curcuminoid extract, hydrolyzed collagen and green teaextract alone and in combination on monolayer cultured normal bovine andhuman osteoarthritic chondrocytes. The in vitro effects for thecombination of the nutraceuticals were significantly effective over theingredients alone in providing beneficial effects on chondrocytesculture in inflammatory conditions. However, the data on combinationstudies show only a physical mixture of the antioxidant green teaextract with collagen peptides. There is no disclosure on any possiblemolecular interactions of green tea extract and collagen peptides or onits solubility, bioavailability, or any synergic effects.

Solid dosage forms for oral delivery of curcuminoids in prior art haveattempted reduction in particle size of curcumin to form suspensions;modification of crystalline form; entrapment of curcumin molecules inliposomes or oil/lipid particles; use of polymer carriers embedded withcurcumin to form dispersions or polymer loaded nanoparticles; and,formation of inclusion compounds withcyclodextrins/phosphatidylcholines, to increase solubility, stabilityand accessibility in or to the gastro-intestinal tract. However, most ofthem are not water soluble, and only dispersible. Their stability inwater and under pH conditions of GI tract is also very poor.

Curcuminoid formulations using various macromolecular carrier forms suchas dietary fiber, proteins, cyclodextrin, and phospholipids etc. areunsuccessful to enhance the bioavailability. These methods failed toprovide completely water soluble curcuminoids in stable formulations inthe pH range from 2 to 7. They also failed in the delivery of bioactivefree curcuminoids over conjugated curcuminoids such as curcuminglucuronides or sulfates. Their half-life in blood was very lowindicating the inability to stay for longer duration at significantlyhigh concentrations.

Proteins like albumin have been used in the formulation of curcuminoids.For protein-based formulations, the bioactive hydrophobic molecules arefirst of all dissolved in organic solvents like DMSO, chloroform,methylene dichloride, acetone etc. and then added to protein solutionfor inclusion in their hydrophobic pockets of 3 dimensional structure.The loading levels of molecules like curcuminoids is also very low andmay provide nano particles of less than 100 nm size in a very low yield.The preparation includes use of toxic organic solvents, the very lowlevels of curcumin loading, low yield, nano form and high cost veryoften remains as a challenge for its usage in food and nutrition field.These becomes critical where phytonutrients are used as foodsupplements, like Sachet or beverages.

Therefore, there exists an acute need for stable, water-soluble, highlyeffective, food-grade biologic compositions and formulations which canprovide better bioavailability and hence more efficacious oral deliveryin various food and pharmaceutical delivery forms. The importance ofsuch compositions and formulations increase when they become food-gradesafe, by avoiding the use of toxic organic solvents, are stable, watersoluble and suitable for oral consumption as and in food and beverages.The water based, water soluble, non-NANO (<100 nm) and stable food-gradeformulations with 10 to 20% (w/w) of bioactive molecules employingfunctional materials such as protein hydrolysates which itself has itsown therapeutic activity when consumed at physiologically relevantdosage.

The present invention overcome these difficulties by providing apeptide-bioactive molecule complex in an encapsulated and stable form ofpeptide networks. Collagen peptides and curcuminoids are primarily usedto explain the present invention.

SUMMARY

A water-soluble formulation comprising peptides from proteinhydrolysates, and hydrophobic bioactive molecules, and method of makingthis formulation are described herein.

One embodiment of the current invention is a water solublepharmaceutical composition comprising: (a) an effective amount of one ormore hydrophobic bioactive molecules; (b) water-soluble peptides derivedfrom protein hydrolysates; and (c) an emulsifying agent, wherein thewater soluble protein hydrolysate peptides form an amorphous andnano-sized (100-1000 nm) non-covalent complex with the hydrophobicbioactive molecules.

In one embodiment, the hydrophobic bioactive molecules for the watersoluble formulation described herein are selected from the groupconsisting of curcuminoids, flavonoids, stilbenes, carotenoids,saponins, terpenes, terpenoids, and chlorophyll. In one embodiment, thehydrophobic bioactive molecules are present at a concentration in therange of 1 to 25% in the formulation disclosed herein.

In one embodiment, the protein hydrolysate peptides are present at aconcentration in the range of 10 to 90% in the composition.

In another embodiment, the composition provides oral administration ofboth the protein hydrolysate peptides and the bioactive moleculestogether in their physiologically relevant dosage without solubility orstability issues.

In one embodiment, the co-administration of hydrophobic bioactivemolecules in the peptide matrix may help synergistic pharmacologicaleffects and better bioavailability of the bioactive molecule.

In one embodiment, the composition described herein is stable atphysiological pH.

In one embodiment, the composition described herein is stable at acidicpH.

In one embodiment, the hydrophobic bioactive molecules are curcuminoids.In one embodiment, the protein hydrolysate peptides are 1000-5000 Da insize.

In one embodiment, the protein hydrolysate peptides are collagenpeptides. In one embodiment, the curcuminoids are complexed with andencapsulated in the collagen peptide matrix.

In one embodiment, the bioactive molecules comprising the formulationexhibit enhanced solubility and stability as compared to unformulatedbioactive molecules. In one embodiment, the pharmaceutical compositiondisclosed herein exhibits increased bioavailability, increasedabsorption and longer half-life of the bioactive molecules after oraladministration to a subject, as compared to unformulated bioactivemolecules.

In one embodiment, the bioactive molecules comprising the complexesdisclosed herein exhibit increased cellular permeability, as compared tounformulated bioactive molecules.

In one embodiment, the formulation disclosed herein exhibits increasedanti-inflammatory effects as compared to unformulated bioactivemolecules.

In one embodiment, the formulation disclosed herein is orallyadministered either as capsules, tablets, softgels, beadlets, liquidsolution, liquid suspension, and liquid emulsion or as powder sachets(drinks) format for enhanced bioavailability of the bioactive molecules.

In one embodiment, it is administered orally at a dose of 100 to 500mg/dose in the form of tablets, capsules or softgel to deliver 25 to 100mg of bioactive molecule in a bioavailable format.

In one embodiment, it is administered orally at a dose of 1000 mg to5000 mg per sachet, once or twice per day, to deliver minimum 100 to 500mg of bioactive molecules per day along with 1 to 10 g ofpeptides/dosage/day.

In one embodiment, the powder form of the formulation disclosed hereinis amorphous.

One embodiment of the current invention is a method of making awater-soluble pharmaceutical composition comprising collagen peptidesand curcumin, wherein the collagen peptides form a water soluble,stable, amorphous and nano-sized non-covalent complex with thecurcuminoids, the method comprising the steps of: (a) micronisation ofcurcuminoids followed by emulsification by homogenisation with anemulsifier; (b) dissolving collagen peptides in water at a concentrationrange of 20 to 95%; (c) complex formation of curcuminoids with thecollagen matrix by sonication with the emulsified curcuminoids from step(a) and the dissolved collagen peptides from step (b) and; (d)encapsulation of the complex from step (c) by making sub-micron sizedmicelle suspension by ultrasound mediated homogenisation; and (e) dryingthe submicron sized micelles from step (d) by spray drying or freezedrying or other low temperature drying techniques.

In one embodiment, the emulsifier is selected from the group consistingof lecithin, polysorbate, propylene glycol, sorbitol, glycerol,polyglycerol esters, Quillaja extract, and sugar esters.

Further scope of applicability of the present invention will becomeapparent from the detailed description given hereinafter. It should beunderstood that the description and the examples, while indicatingembodiments of the invention, are given by way of illustration only,since various changes and modifications within the spirit and scope ofthe invention will become apparent to those skilled in the art from thedescription. It is to be understood that the description and examplesare explanatory and representative of the invention and in no way limitor restrict the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows fluorescence analysis of the CCL complex. C95—Curcuminoidswith 95% purity isolated from turmeric rhizomes;CCL—Curcuminoids-collagen complex.

FIG. 2 shows Ultraviolet scan data of the CCL complex. C95—Curcuminoidswith 95% purity isolated from turmeric rhizomes;CCL—Curcuminoids-collagen complex; CP-collagen peptide.

FIG. 3 shows FTIR data for the CCL complex. C95—Curcuminoids with 95%purity isolated from turmeric rhizomes; CCL—Curcuminoids-collagencomplex; CP-collagen peptide.

FIG. 4A shows ¹H NMR data of C95—Curcuminoids with 95% purity isolatedfrom turmeric rhizomes.

FIG. 4B shows ¹H NMR data of CP-collagen peptide.

FIG. 4C shows ¹H NMR data of CCL—Curcuminoids-collagen complex.

FIG. 5A shows ¹³C NMR data of C95—Curcuminoids with 95% purity isolatedfrom turmeric rhizomes.

FIG. 5B shows ¹³C NMR data of CP-collagen peptide.

FIG. 5C shows ¹³C NMR data of CCL—Curcuminoids-collagen complex.

FIG. 6 shows DSC analysis of the CCL complex. C95—Curcuminoids with 95%purity isolated from turmeric rhizomes; CCL-Curcuminoids-collagencomplex; CP-collagen peptide.

FIG. 7 shows SEM analysis of the CCL complex. C95—Curcuminoids with 95%purity isolated from turmeric rhizomes; CCL-Curcuminoids-collagencomplex; CP-collagen peptide.

FIG. 8 shows particle size analysis of CCL solution (A), and particlesize analysis of CCL solution filtered through a 420 nm filter (B).

FIG. 9 shows TEM data. CP—Collagen peptide and CCL—Curcuminoid-collagencomplex.

FIG. 10 shows photographs of CCL and C95 water solutions. A. C95 inwater, B. CCL in water and C. CCL in water after filtration through 200nm filter.

FIG. 11 shows the plasma concentration of curcuminoids when supplementedwith unformulated curcumin 95%, water dispersible physical mixture ofcurcumin and collagen peptides, and curcumin-collagen complex (CCL) incapsule and sachet format.

FIG. 12 shows cellular permeability of CCL in comparison withunformulated curcumin 95.

FIG. 13 shows cell proliferation inhibitory effect of CCL in SAS celllines in comparison with unformulated curcumin 95.

FIG. 14 shows cytotoxicity of CCL investigated by flow cytometry incomparison with unformulated curcumin 95.

FIG. 15 shows anti-inflammatory effect of CCL in comparison withunformulated curcumin 95.

DETAILED DESCRIPTION

The invention is not limited to various embodiments given in thisspecification. The terms used in this specification generally have theirordinary meanings in the art, within the context of the invention, andin the specific context where each term is used. Unless otherwisedefined, all technical and scientific terms used herein have the samemeaning as commonly understood by one of skill in the art to which thisinvention pertains. Certain terms used herein are described below, orelsewhere in the specification to provide additional guidance to apractitioner regarding description of the invention. In case ofconflict, the present document, including definitions will control.

As used herein, the terms ‘comprising’, ‘including’, ‘having’,‘containing’, involving’ and the like are to be understood to be openended, i.e., to mean including but not limited to.

As used herein, the terms ‘curcumin’ and ‘curcuminoids’ are usedinterchangeably and indicate the biologic phenols obtained from theCurcuma plant. In the present application, ‘curcuminoids’ refers to thestandard curcumin with not less than 95% purity isolated from turmericrhizomes by solvent extraction with a composition of curcumin 72 to 80%w/w, DMC (demethoxycurcumin) 12 to 15% w/w and BDMC(bisdemethoxycurcumin) 3 to 5% w/w.

As used herein, the term “unformulated bioactive molecules” refers tohydrophobic biomolecules not in complex with any carrier molecules ormatrix. It refers to bare bioactive molecules, without any extraneouslyadded carrier molecules.

The term “unformulated curcuminoids” refers to curcuminoids not incomplex with any carrier or delivery molecules or matrix. It includesthe curcuminoids that are isolated from the turmeric rhizome and notbound to any other extraneously added carrier molecules.

As used herein, the term “effective concentration”, “effective amount”means a concentration of the formulations described herein which cangenerate the desired effect in the subjects/patients, without leading toundesired levels of side effects. The effective concentrations of theformulations disclosed herein are given the various embodiments herein,as dosages of the formulation in different delivery formats.

As used herein the term “C95” refers to Curcuminoids with 95% purityisolated from turmeric rhizomes; “CP” refers to collagen peptide, “CCL”refers to curcuminoids-collagen complex.

The current invention provides water-soluble formulations comprisingpeptides from protein hydrolysates, and hydrophobic bioactive molecules,and method of making this formulation.

One embodiment of the current invention is a water solublepharmaceutical composition comprising: (a) an effective amount of one ormore hydrophobic bioactive molecules; (b) water-soluble peptides derivedfrom protein hydrolysates; and (c) an emulsifying agent, wherein thewater soluble protein hydrolysate peptides form an amorphous andnano-sized non-covalent complex with the hydrophobic bioactive moleculesand acts as a matrix for its effective encapsulation in solution.

In one embodiment, the hydrophobic bioactive molecules for the watersoluble formulation described herein are selected from the groupconsisting of curcuminoids, flavonoids, stilbenes, carotenoids,saponins, terpenes, terpenoids, and chlorophyll. In one embodiment, thehydrophobic bioactive molecules are present at a concentration in therange of 1 to 25% in the formulation disclosed herein.

In one embodiment, the protein hydrolysate peptides are present at aconcentration in the range of 10 to 90% in the composition. In anotherembodiment, such peptides have nutritional properties and are derivedfrom food proteins.

In one embodiment, the hydrophobic bioactive molecules are curcuminoids.In one embodiment, the protein hydrolysate peptides are 1000-5000 Da insize.

In one embodiment, the protein hydrolysate peptides are collagenpeptides. In one embodiment, the curcuminoids are complexed with andencapsulated in the collagen peptide matrix, where peptides act as aprotective coat of the water soluble bioactive micelle or liposomes orphytosomes formed between the emulsifiers and hydrophobic bioactivemolecules.

In another embodiment, peptides provides a noodle like structure inwhich the micelles or liposomes or water miscible particles of thehydrophobic molecules are trapped and protected.

In one embodiment, it exhibits enhanced solubility and stability ascompared to unformulated bioactive molecules. In another embodiment, thepeptide network of the formulation swells in the gastrointestinal (GI)fluid and is acted upon by the GI enzymes to convert to smaller peptidesand amino acids and the leached hydrophobic molecules get easilyabsorbed, thus enhancing bioavailability.

In one embodiment, the pharmaceutical composition disclosed hereinexhibits increased bioavailability, increased absorption and longerhalf-life of the bioactive molecules after oral administration to asubject, as compared to unformulated bioactive molecules.

In one embodiment, the composition exhibits increased cellularpermeability, as compared to unformulated bioactive molecules.

In one embodiment, the composition disclosed herein exhibits increasedanti-inflammatory and antioxidant effects as compared to unformulatedbioactive molecules.

In one embodiment, the composition disclosed herein is orallyadministered either as capsules, tablets, softgels, beadlets, liquidsolution, liquid suspension, and liquid emulsion or as powder sachets(drinks) for enhanced bioavailability of the bioactive molecules.

In one embodiment, it is administered orally at a dose of 100 to 500mg/dose in the form of tablets, capsules or softgel to deliver 25 to 100mg of bioactive phytonutrient molecule in a bioavailable format.

In one embodiment, it is administered orally at a dose of 1000 mg to5000 mg per sachet, once or twice a day, to deliver minimum 100 to 500mg of bioactive molecules per day along with 1 to 10 g ofpeptides/dosage/day. In one embodiment, wherein the powder form of theformulation disclosed herein is amorphous.

In one embodiment, the method of making a water-soluble pharmaceuticalcomposition comprising collagen peptides and curcumin, wherein thecollagen peptides form a water soluble, stable, amorphous and nano-sizednon-covalent complex with the curcuminoids, the method comprising thesteps of: (a) micronisation of curcuminoids followed by emulsificationby homogenisation with an emulsifier; (b) dissolving collagen peptidesin water at a concentration range of 20 to 95%; (c) complex formation ofcurcuminoids with the collagen matrix by sonication with the emulsifiedcurcuminoids from step (a) and the dissolved collagen peptides from step(b); (d) encapsulation of the complex from step (c) by making sub-micronsized micelle suspension by ultrasound mediated homogenisation; and (e)drying the submicron sized micelles from step (d) by spray drying orfreeze drying.

In one embodiment, the emulsifier is selected from the group consistingof lecithin, polysorbate, propylene glycol, sorbitol, glycerol,polyglycerol esters, Quillaja extract, and sugar esters.

One embodiment of the pharmaceutical composition made by the methoddescribed above.

Collagen peptides support the integrity, elasticity, regeneration andstrength of the connective tissues including skin, bones, cartilage,ligaments and tendons provide beneficial health effects to skin andbones, especially in improving joint mobility, ameliorating joint painand the like associated with osteoarthritis.

In one embodiment, the present invention uses collagen hydrolysates orcollagen peptides to prepare the complexes with hydrophobic bioactivemolecules. In one embodiment, collagen peptides contains 19 amino acids.In one embodiment, the collagen hydrolysate or collagen peptides areprepared by enzymatic hydrolysis. In one embodiment, the collagenpeptides used in the current invention are water soluble. Creatingdelivery forms with short peptides like collagen in solution is verydifficult due to the fact that the short soluble peptides like collagenmolecules does not have suitable conformation and 3dimensional structurewith suitable hydrophobic pockets for engulfing hydrophobic moleculeslike curcuminoids.

In one embodiment, the short length water soluble collagen peptides usedherein have molecular weight ranging from 2000 to 5000 Da. The watersoluble peptides have a molecular weight ranging from 1000 to 20000 Da,more particularly 1000 to 5000 Da and more preferably 1000 to 3000 Da,obtained from the enzymatic hydrolysis of collagen, a high molecularweight animal protein of ˜300 kDa.

In one embodiment, the short collagen peptides act as carrier or vehiclefor hydrophobic and poorly bioavailable small bioactive molecules likecurcuminoids to convert them into water soluble, easily dispersiblecurcuminoids with enhanced bioavailability when delivered orally. Theseproperties are achieved by a complex formulation where by curcuminoidsare associated with collagen peptides in a non-covalent, complexformation.

In one embodiment, these short collagen peptides successfully formcomplexes without the formation of any chemical cross links.

In one embodiment, the complexes disclosed herein possess interaction ofcurcuminoids with collagen peptides and also create strong Van der Waalsforce of attraction such as hydrogen bonding between collagen peptidemolecules and curcuminoids.

In one embodiment, the strong interaction between emulsifiedcurcuminoids and short collagen peptides results in a protective coatingof the curcuminoids micelles or liposomes or phytosomes with collagenpeptides to attain better stability in vivo. The interaction alsoresults in an increase in the water solubility of hydrophobiccurcuminoids, increased bioavailability of curcuminoids, and henceimproved therapeutic efficacy.

In one embodiment, the present invention of collagen peptide-curcumincomplex claim superiority a mere physical mixture or water dispersiblephysical mixture formulated by mixing the collagen peptides andcurcuminoids, in terms of its solubility, stability, bioavailability andbio-efficacy. The physical mixture results in a water-insoluble,indispersible (not-dispersible) mixture of curcumin that settles to thebottom within few seconds thereby making its consumption difficult. Sucha mixture will have low bioavailability without any synergistic effect,and will lack in sensory appeal necessary for an edible product.

In one embodiment, compositions and formulations comprising the uniquecollagen peptide-curcuminoid complexes disclosed herein providesignificantly high levels of bioavailable curcuminoids that is watersoluble and stable at physiological and acidic pH along with significantanti-inflammatory effects offered by collagen peptides.

In one embodiment, the collagen peptide-curcuminoid complex is in awater soluble, stable, solid dosage form having a concentration of 100to 250 mg curcuminoids in every 3 g of collagen peptides, preferably in2 g collagen peptides, or more preferably in 1 g collagen peptidessuitable for oral consumption which renders collagen peptides andcurcuminoids bioavailable for enhanced efficacy on skin, bone, joint andgastrointestinal health.

In one embodiment, compositions and formulations comprising the collagenpeptide-curcuminoid complexes can be conveniently made to delivereffective dose of curcuminoids orally. In one embodiment, the collagenpeptide-curcuminoid complex in the composition disclosed herein leads toincrease in the water solubility of curcuminoids and their in vivostability.

In one embodiment, the collagen-curcumin compositions and formulationsexhibit better absorption and bio-availability when consumed orally.

In one embodiment, the collagen peptide-curcuminoid complex disclosedherein can be formulated for oral consumption in solid and liquid doseforms such as pills, capsules, tablets, soft-gels, emulsions, solutions,syrups and the like for delivering effective dose of collagen peptidesand curcuminoids orally.

In an embodiment, the collagen peptide-curcuminoid complex disclosedherein can be formulated into food and drink edible compositions likepowder mixes, granules, ready-to-drink liquids, beverage powders, drinkand dairy mixes, confectionaries, and dietary supplements to name a few,for delivering effective dose of collagen and curcuminoids orally.

In an embodiment, powder formulations of curcuminoid-collagen peptidecomplexes suitable for supplementation at 1 to 10 g/serving of collagenpeptides and 250 to 500 mg curcuminoids/serving is achieved as singleready to drink water soluble powder. The formulation not only providesbioavailable forms of bioactive molecules like curcuminoids, but alsoprovides therapeutically significant dosage of peptides like collagenpeptides together for synergic effect.

In another embodiment, the oral formulation of curcuminoid collagenpeptide complexes suitable for supplementation as a sachet/to consumealong with water, milk or yogurt, soups, ice creams, etc.

Although the present invention has been illustrated and described hereinwith reference to the embodiments and examples disclosed herein, it willbe readily apparent to those skilled in the art that other embodimentsand examples may perform similar functions and/or achieve like results.All such equivalent embodiments and examples are within the spirit andscope of the present invention, are contemplated thereby, and areintended to be covered by claims put forth in the application.

EXAMPLES

The examples below illustrate the present invention and arenon-limiting. The below exemplify representative embodiments of theinvention, where all parts, proportions and percentages are by weightunless otherwise indicated. All fine chemicals, reagents, solvents andthe like were commercially procured.

Collagen peptides from bovine, chicken and fish were purchased fromNitta Gelatin India Limited, Kochi, Kerala State, India. Phospholipidswere purchased from Lipoid AG, Steinhausen, Switzerland. Curcumin 95%was taken from the quality assurance laboratory of Akay Flavours andAromatics Pvt Ltd., Kochi, Kerala, India.

Example 1: Preparation of Curcumin-Collagen Complex: Preparation ofCurcuminoid Suspension

About 9.2 g of commercially procured curcumin with not less than 90%purity was mixed with 4.5 g of MCT oil and with 4.5 g of glycerine andheated for 30 min to get a uniform mass. 9 g of lecithin having aphosphatidyl choline content of 20 to 90%, most preferably 25 to 45%,was then dissolved in 50 mL of ethanol/water mixture by homogenizationor sonication. The water dispersible curcumin mass was then added slowlyto ethanol/water by sonication and further suspended in 200 mL waterwith high power ultrasound (1000 KW) as pulses of 1 to 3 seconds orhomogenization. Ethanol was then evaporated under vacuum to obtain awater suspension of curcuminoids having an approximate particle size notmore than 2 μm.

Preparation of Collagen Peptide-Curcuminoid Complex:

70 g of commercially obtained collagen peptides was separately dissolvedin 400 mL water by homogenization. The solution was slowly mixed withthe curcuminoids suspension (275 mL) by high shear mixing, optionallyheating. The solution was homogenized and then spray dried or freezedried to obtain free-flowing, water-soluble, stable, collagenpeptide-curcuminoids complex having total curcuminoids content of 10.2%.

Alternatively emulsifiers including lecithin, polysorbate, propyleneglycol, sorbitol, glycerol, and polyglycerol esters, Quillaja extract,or sugar esters, can be used with and without lecithin to make collagenpeptide-curcuminoids complexes.

Fatty oils including but not limited to sunflower oil, coconut oil,polyunsaturated fatty oils, olive oil or fish oil may be used instead ofMCT oil.

The composition of ethanol/water can vary from 70:30 v/v to 95:5 v/v.

Temperature during formulation can vary from 50 to 80° C., depending onthe stability of the bioactive substance.

Peptides can be derived from any food-grade proteins having watersolubility and molecular weight ranging from 1000 to 10000, preferably1000 to 5000, more preferably 1000 to 3000 Da.

The amount of curcumin can vary from 1 to 25% to producecurcumin-collagen complex with varying levels of curcuminoids contentpreferably less than 20%.

Example 2: Preparation of Curcumin-Fava Peptide Complex

Curcumin-fava peptide complex can be used by using the similar processdisclosed in Example-1. The fava protein hydrolysate (i.e. fava peptidewith molecular weight of around 3000 Da) and polysorbate 3 g was usedinstead of Glycerol.

Example 3: Preparation of Collagen Peptide-Gingerol Complex

Gingerol is the bioactive molecule from Ginger and is water insoluble.

Supercritical ginger extract containing 40% of gingerols was used forthe formulation. It is obtained as an oil soluble paste form(Oleoresin). No oils were used in this formulation as specified inexample 1. Briefly, about 18 g of oleoresin was mixed with and 18 g oflecithin with heating in ethanol/water medium. The ethanol was thenevaporated and emulsified in water containing 3% Quillaja extract. Thewater-soluble form is then mixed with the collagen peptide withmolecular weight 5000 and again sonicated. The uniform solution is thenkept for 2 h. The supernatant solution is separated and freeze dried tofine water-soluble powder. Gingerol content was 6.3%.

Example 4: Characterisation of Collagen Peptide-Curcuminoids Complex

Spectroscopic techniques such as Fluorescence, Ultraviolet, Infrared,and NMR were used to confirm the non-covalent complex formation ofcurcuminoids with peptides. The shifts in the characteristic peaks dueto hydrogen bonding between the peptide bonds, amino acid side chainsand curcuminoids were confirmed. Proton (¹H) and ¹³C NMR studies showthe inclusion of curcuminoids in the collagen network. Powder X-raydiffraction studies and differential scanning calorimetric confirms theamorphous nature of the complex whilst electron microscopic studiesprovide information on particle size, surface morphology and moleculararrangements in the complex.

Solid-state Fourier-transformed infrared spectra (FTIR) was recorded onShimadzu spectrophotometer 8700 using potassium bromide pellets,prepared by compressing the powder at 20 psi for 10 min on a KBr press(Shimadzu Analytical Pvt. Ltd., Mumbai, India). The spectra were scannedover the wave number range of 3600-400 cm⁻¹. Thermogram was recordedusing a differential scanning calorimeter (DSC) (Mettler-Toledo IndiaPvt. Ltd., Mumbai, India), by heating the samples (3-5 mg) in thealuminum crimp pan at a rate of 10° C./min from 30 to 300° C. undernitrogen atmosphere. Powder X-ray diffraction studies (PXRD) wereperformed on a Bruker D8 Advance instrument: target Cu, k—1.54 A°,filter —Ni, voltage 40 kV, time constant 5 min/s; scanning rate 1°C./min (Bruker AXS GmbH, Karlsruhe, Germany). Scanning electronmicroscopic analysis (SEM) was done on SEM Jeol 6390 LA equipment (JEOLLtd., Tokyo, Japan).

The enhanced bioavailability of curcuminoids presented in the form ofcollagen peptide-curcuminoid complexes was confirmed by measuring theplasma concentrations over 24 h time period as compared to unformulatedcurcuminoids with 95% purity (C95).

Example 4A: Fluorescence Analysis

The fluorescence analysis (FIG. 1) shows that, upon complexation CCL hasan increased intensity of absorption. Curcumin exhibited an emissionspectra max at 580 nm which on complex formation with collagen,decreased to 505 nm, indicating the inclusion of curcuminoids in ahydrophobic environment with hydrophobic interactions. Hence the blueshift in the complex indicates higher energy requirement for theexcitation of molecule which further supports a stronger interaction(complexation) between the curcuminoids and peptides.

Example 4B: Ultraviolet/Visible Spectroscopy Analysis

The Ultraviolet/Visible spectroscopy (FIG. 2) of C95, CP and CCLindicated an interaction between the curcumin moiety and the collagen inCCL. The absorption maxima of curcumin has shifted from 420 nm to 408nm, a blue shift. The absorption peak of curcumin at 420 nm has alsobeen altered to a hill like plot spread out from 190 to 500 nm in CCL.This is due to the influence of the peptide which along with thecurcumin exhibits uniform UV absorption, a multipoint attraction where awide range of chromospheres are available.

Example 4C: Fourier Transform Infrared Spectroscopy (FTIR)

Since, the complexation reaction was performed in water at almostneutral pH, the curcumin exists in an equilibrium between the keto andenol forms. In the keto form, the oxygen atom attached to the carbonylgroup has a higher electronegativity due to the adjacent αβunsaturation. This electronegativity of carbonyl oxygen attracts thelone pairs of the amide bond in the peptide chains, leading tointermolecular hydrogen bonding. This is evident from the shift of the1647 cm⁻¹ of the amide bond to 1631 cm⁻¹ in CCL. The small peaks due tothe stretching vibrations of enol-carbonyl groups at 1625 cm⁻¹ and 1599cm⁻¹ were widened and observed as a shoulder of 1631 cm⁻¹ in CCL (FIG.3). The peaks at 1426 cm⁻¹ in curcumin due to its C═C vibration isshifted to 1405 cm⁻¹ in CCL, indicating the presence of keto form andits hydrogen bonding. The absorption peak at 3510 cm⁻¹, which is thetypical peak of the —OH group of the phenolic structure and theabsorption peak at 3410 cm⁻¹ which is the typical peak of the normalamine group has fused together and got shifted to 3443 cm⁻¹ indicatingan interaction between groups. Thus, the two —OH groups in the aromaticrings and the —OH and keto form of the keto-enolic form in curcumin hasinteractions with peptide bonds and terminal groups of peptides. Yetanother possible reason for the shifts may be the hydrophobicinteractions due to the inclusion of the curcuminoids in the hydrophobicpockets formed by the entanglement of random coiled peptides to formnoodle-like structure for CCL.

Example 4D: NMR Studies

NMR studies revealed the extent of complexation in CCL. The ¹H NMR data(FIG. 4A, B, C) of curcumin indicated the presence of a high shieldedhydrogen atom (hydrogen of the enol form) with chemical shift at 16.053ppm. Due to low shielding effect, the —OCH₃ group in the aromatic ringof curcumin exhibited sextet at 3.949 ppm. Upon compexation, the highershielding effect of the enol form was lost. Similarly, the sextet at3.949 ppm got shifted towards 3.629 ppm which indicated a furtherlowering of the shielding effect. This is due to the association of thepeptide fractions at the —OH group adjacent to it. The hydrogensplitting raised from 6 to 7.55. 3.629 ppm which indicated a furtherlowering of the shielding effect. This is due to the association of thepeptide fractions at the —OH group adjacent to it. The hydrogensplitting raised from 6 to 7.55.

As per the ¹³C NMR data (FIG. 5A, B, C), the high shielded carbon of theketo-enolic form in curcumin was observed at 184.521 ppm. Upon CCLformation, the chemical shift lowered to 172.366 ppm. Similarly, thecarbon atom of the —OCH₃ group, which was observed at 57.222 ppm incurcumin got further lowered to 46.085 ppm which indicated lowering ofshielding effect. This too indicates the non-covalent force ofattraction that may have formed in between the aromatic —OH group andthe terminal —NH₂ of the peptide.

Example 4E: Differential Scanning Calorimeter (DSC) Analysis

DSC analysis (FIG. 6) analysis exhibited a sharp endothermic shift at175.74° C. for curcumin due to its melting. The collagen peptideexhibited a slight endothermic shift at 74.60° C. and 194.23° C. Uponcomplexation, the CCL exhibited a shift at 71.77° C., 179.49° C. and196.33° C., indicating an adduct/bonding between the two entities.However, no sharp shift was observed, due to the amorphous nature ofCCL.

Example 4F. Scanning Electron Microscopy (SEM)

The scanning electron microscopy analysis of the powder samples (SEM)(FIG. 7) indicated the change in surface nature of the curcuminoids andcollagen peptides. The curcuminoids were crystalline as evident from theneedle shapes. Collagen peptide on the other hand is amorphous and formsspherical forms. The CCL complex is also spherical with smooth surfaceindicating the encapsulation of crystalline curcuminoids into thecollagen network and its change into amorphous form.

Example 5: Particle Size and Transmission Electron Microscopy (TEM)Analysis of CCL in Water Example 5A: Particle Size Analysis

Particle size/zeta potential of CCL in solution was analyzed by adynamic light scattering Mastersize 3000 equipment of MelvernPananalytical, USA. About 100 mg of curcuminoid-collagen complex wassuspended in 10 mL distilled water for 1 h and shaken in a rotatoryshaker. The supernatant solution was subjected to particle sizeanalysis, which indicated two fractions having a mean particle size ofless than 200 nm (150 to 200 nm) and relatively bigger particles ofaround 700 nm (600 to 700 nm). The solution was also filtered through a420 nm Nylon filter and again subjected to particle size analysis. Theparticles were of around 176 nm (FIG. 8A, 8B).

Example 5B: TEM Studies

The morphology of particles in solution was visualized undertransmission electron microscopy (JEM-2100, JEOL, Tokyo, Japan). Aqueoussolution of curcuminoid-collagen complex was placed (20 μL) on a coppergrid and allowed to dry at room temperature. After staining with 2%phosphotungstic acid, the thin film formed was subjected for TEMobservation. TEM indicated the agglomeration of curcumin with collagenpeptides, with strong association of both to observe as a singlemolecular solution (FIG. 9).

Example 6: Water Solubility and Stability of CollagenPeptide-Curcuminoid Complex

About 20 mg of curcumin 95% which contains 19 mg of pure curcumin byHPLC was weighed into a 100 mL standard flask and marked as C95. 100 mgof CCL which contains 19 mg of pure curcumin by HPLC was weighed intoanother 100 mL standard flask and marked as CCL. Both were made up tothe volume using HPLC grade water and were sonicated in a bath sonicatorfor 5 min. It was centrifuged and the supernatant was subjected to HPLCanalysis to measure the dissolved curcumin content. It is observed thatcurcuminoid content in C95 had 14 ng/mL and that in CCL was 2040 μg/mL,indicating enhancement in solubility of curcuminoids (FIG. 10).

Example 7: Storage Stability of Collagen Peptide-Curcuminoid Complex

Storage stability studies were carried out as per the guidelines ofInternational Conference on Harmonization (ICH) of technicalrequirements for registration of pharmaceuticals for human use (ICH,2003). Briefly, the sample packets (10 g) of curcuminoid-collagencomplex was sealed in a double layered polyethylene bags and wereincubated at 40±2° C. and 70±5% relative humidity for a period of 6months in a stability chamber (Remi, Mumbai, India). The samples werewithdrawn at 0, 1, 2, 3, and 6 months and subjected to analysis forvarious physical and chemical parameters. Curcuminoids content, moisturecontent and microbial parameters comprising total plate count, yeast andmold, Escherichia coli, Salmonella and coliforms were measured, resultsare shown in Table 1.

TABLE 1 Parameters Specification 0 month 3 month 6 month Appearance Freeflowing Complies Complies Complies powder Identification HPLC CompliesComplies Complies Colour Yellowish Complies Complies Complies orangeCurcumin >20% 20.68% 20.35% 20.19% content Density 0.4-0.8 g/mL 0.42g/mL 0.42 g/mL 0.42 g/mL Microbiology US-FDA (BAM) Total plate <3000cfu/g 100 cfu/g 110 cfu/g 100 cfu/g count Yeast & Mold <100 cfu/g <10cfu/g <10 cfu/g <10 cfu/g Coliforms <3 MPN/g <3 MPN/g <3 MPN/g <3 MPN/gEscherichia Absent/g   Absent/g   Absent/g   Absent/g   coli SalmonellaAbsent/25 g Absent/25 g Absent/25 g Absent/25 g

Example 8: Bioavailability Studies

The enhanced bioavailability of curcuminoids presented in the form ofcollagen peptide-curcuminoid complexes is confirmed by measuring theplasma concentration of curcuminoids over 12 h time period as comparedto curcuminoids 95%. Human studies were conducted in accordance with theclinical research guidelines Healthy adult human volunteers (5 males and3 females; aged between 24 and 46 years), who were not under anymedication or dietary supplements were selected for the study. Subjectsare assigned with a three digit unique randomization code.Ultraperformance liquid chromatography coupled with electrosprayionization tandem mass spectrometer (UPLC-ESI-MS/MS) (6460 Massspectrometer, Agilent India Pvt Ltd, Bangalore, India) was employed forthe analysis of curcuminoids in plasma or serum or whole blood collectedafter various post-administration time intervals (0, 1, 3, 5, 8 and 12h).

Extraction efficiency of curcuminoids from plasma was confirmed byspiking 10 μg/mL of standard solution in 1 mL of blank plasma followedby analysis. Retention time was confirmed by 10 repeated analyses at 20μg/mL level on the same column under identical conditions. Measurementof the contents in plasma was validated by spiking standard the activesin plasma at 10 and 20 μg/mL concentrations. Extraction efficiency fromplasma was 94% with a linear response of R² value of 0.998. The identitywas established by analytical standards and by multiple reactionmonitoring (MRM) of their MS/MS spectra in mass spectrometry.

In a typical protocol followed in the present study, each volunteer wasfirst given one capsule of 500 mg (500 mg×1) of unformulatedcurcuminoids 95% or Curcuminoid-collagen complex (500 mg ofCurcuminoid-collagen complex containing 20% curcuminoids as capsules);blood samples were withdrawn; plasma was separated and frozen at −20° C.till analysis. After 1 week, the subjects were cross-covered andprovided with either one capsule of either 500 mg ofCurcuminoid-collagen complex containing 20% curcuminoids or unformulatedcurcuminoid 95%. The protocol of collection of blood and plasma wasrepeated exactly the same as above. Plasma concentration verses timeplot was constructed for the detailed analysis of pharmacokinetics ofcurcuminoids and their respective formulations to deduce bioavailabilityor relative absorption.

The same procedure was also repeated with the supplementation of 3 g ofcollagen-curcuminoids complex containing 8.5% of curcuminoids as aready-to-drink sachet. Here the powder complex was dissolved in 200 mLof drinking water allowed to drink in such a way that one sachetprovides 250 mg of curcuminoids in a soluble form. The blood and plasmasamples at various post-administration time points was collected andsubjects to LC-MS/MS analysis as before.

Pharmacokinetic parameters like the maximum curcumin concentration inthe blood (C_(max)), the time taken to reach the maximum concentration(T_(max)) and concentration of curcumin in blood after 12 hr (C¹²_(max)) were analysed. It can be seen that the curcuminoids in the newformulation absorbs almost 53 times for in Sachet form and 36 times incapsule form more than the normal curcumin, on an average. Moreover,T_(max) for curcumin-collagen complex was 4.2 hr for Sachet and 7 hr forin capsule administration as compared to the normal curcumin.

FIG. 11 shows the plasma concentration verses time graph forcurcuminoids when supplemented with curcumin 95%, water dispersiblephysical mixture of curcumin and collagen peptides, andcurcumin-collagen complex in capsule and sachet format. The table 2provides the pharmacokinetic parameters indicating the higherabsorption, bioavailability and longer duration of existence in theblood when curcumin-collagen was consumed as compared to the curcuminalone or its physical mixture with collagen peptides.

TABLE 2 Cmax Tmax T½ Sample Dosage (ng/mL) (hr) (hr) AUC Folds CCLCapsule 500 mg 74.08 5 7 348.4 36.7899 CCL Sachet 3000 mg 118.84 3 4.2506.8 53.5164 WD Physical 500 mg 10.25 0.5 0.9 16.69 1.76241 mix*Standard 500 mg 4.35 0.5 0.7 9.47 1 Curcumin *WD Physical mix—waterdispersible physical mixture of curcumin and collagen peptides

Example 9: Cell Permeability of CCL

Cell permeability using Inverted Fluorescence Microscope. Fluorescentmicroscopy was employed to examine the cellular uptake and distributionchanges of LFCSNs into HepG2 cells. The HepG2 cells approximately(1×10⁵) were cultured in a six well plate in a medium made up of EMEMand treated with of LFCSNs for 24 and 48 h with technical triplicates,untreated control and treated cells were observed under invertedmicroscope. For each experiment, nuclei from 10 random fields of wellwere examined at 200× magnification (FIG. 12). From the result (FIG.12), cellular uptake of CCL is clear as compared to C95.

Example 10: Cell Toxicity of CCL Example 10A: MTT Assay for CellProliferation

The MTT assay is used to measure cellular metabolic activity as anindicator of cell viability, proliferation and cytotoxicity. Thiscolorimetric assay is based on the reduction of a yellow tetrazoliumsalt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide orMTT) to purple formazan crystals by metabolically active cells. Theproliferation study was conducted in SAS cell line. FIG. 13 shows therelative cellular toxicity of CCL in comparison with C95 in SAS celllines.

Example 10B: Cytotoxicity Assay Using PI FACS

Flow cytometry provides a rapid and reliable method to quantify viablecells in a cell suspension. Live cells have intact membranes thatexclude a variety of dyes that easily penetrate the damaged, permeablemembranes of non-viable cells. Propidium iodide (PI) is a membraneimpermeant dye that is generally excluded from viable cells. It binds todouble stranded DNA by intercalating between base pairs. PI is excitedat 488 nm and, with a relatively large Stokes shift, emits at a maximumwavelength of 617 nm. In this study 25 ug/mL concentration of C95 inDMSO and CCL in water were used. FIG. 14 shows the flow cytometery datafor CCL vs C95.

Example 11: Anti-Inflammatory Effect of CCL

Carrageenan-Induced Mouse Paw Edema Model: Male Swiss albino mice weredivided into four groups. First group was kept as vehicle control;second group treated with unformulated curcumin at a dose of 50 mg/kgb·wt; third group was treated with CCL at a dose of 50 mg/kg b·wt. Fifthgroup was treated with standard drug diclofenac at a dose of 25 mg/kgb·wt. The carrageenan-induced mouse hind paw edema test was employedusing the method described anteriorly with some modifications. Briefly,after administration for 60 min, each animal except the intact controlwas injected with 25 μL of 1% freshly prepared carrageenan suspension atthe plantar side of right hind paw. The paw edema values were evaluatedbefore as the basal volume (tC₀) and 1, 2, 3, 4, 5, or 6 h as thepathological volume (tC_(n)) after carrageenan injection by the MK101CMPpaleothermometer (Muromachi Kikai Co., Ltd., Japan). The percentagedegree of swelling and inhibition of paw edema were calculated using thefollowing formulas (FIG. 15):

$\text{\%~~inhibition}{= \frac{\left( {{tC}_{n} - {tC}_{0}} \right) - {\left( {{tT}_{n} - {Tt}_{0}} \right) \times 100}}{\left( {{tC}_{n} - {tC}_{0}} \right)}}$

Where, tCn=paw thickness at particular time point of control animal;tC₀=paw thick ness of control animals before induction;tT_(n)=paw thickness at particular time point of treated animal; andTt₀=paw thickness of treated animals before induction

It is observed that CCL has offered higher inhibition of paw edemacompared to unformulated curcumin and collagen peptide (FIG. 15).

Example 12

Delivery forms comprising collagen peptide-curcuminoid complexcompositions comprising the collagen peptide-curcuminoid complexesformulated as capsules, tablets, soft-gels, powders, ready-to-consumemixes, granules, liquids for consumption as such, in aqueous beverages,incorporated in dairy products, candies like gummies, milk powder,protein shakes etc., demonstrated the stability and increasedbioavailability of various dosage forms at 100 to 500 mg/serving.

1. A water soluble pharmaceutical composition comprising: a) aneffective amount of one or more hydrophobic bioactive molecules; b)water-soluble peptides derived from protein hydrolysates; and c) anemulsifying agent; wherein the water soluble protein hydrolysatepeptides form an amorphous, nano-sized non-covalent complex with thehydrophobic bioactive molecules.
 2. The pharmaceutical composition ofclaim 1, wherein the hydrophobic bioactive molecules are selected fromthe group consisting of curcuminoids, gingerol, flavonoids, stilbenes,carotenoids, saponins, terpenes, terpenoids, and chlorophyll.
 3. Thepharmaceutical composition of claim 1, wherein the hydrophobic bioactivemolecules are curcuminoids.
 4. The pharmaceutical composition of claim1, wherein the hydrophobic bioactive molecules are present at aconcentration in the range of 1 to 25% in the composition.
 5. Thepharmaceutical composition of claim 1, wherein the peptides are presentat a concentration in the range of 10 to 90% in the composition.
 6. Thepharmaceutical composition of claim 1, wherein the protein hydrolysatepeptides are 1000-5000 Da in size.
 7. The pharmaceutical composition ofclaim 1, wherein the emulsifier is selected from the group consisting oflecithin, polysorbate, propylene glycol, sorbitol, glycerol,polyglycerol esters, Quillaja extract, and sugar esters.
 8. Thepharmaceutical composition of claim 1, wherein the protein hydrolysatepeptides are collagen peptides.
 9. The pharmaceutical composition ofclaim 1, wherein the curcuminoids are complexed and encapsulated in thecollagen peptide matrix.
 10. The pharmaceutical composition of claim 1,wherein the bioactive molecules comprising the composition exhibitenhanced solubility and stability as compared to unformulated bioactivemolecules.
 11. The pharmaceutical composition of claim 1, wherein thebioactive molecules comprising the composition exhibit increasedbioavailability, increased absorption and longer half-life of thebioactive molecules after oral administration to a subject, as comparedto unformulated bioactive molecules.
 12. The pharmaceutical compositionof claim 1, wherein the bioactive molecules comprising the compositionexhibit increased cellular permeability, as compared to unformulatedbioactive molecules.
 13. The pharmaceutical composition of claim 1,wherein it exhibits enhanced anti-inflammatory as compared tounformulated bioactive molecules.
 14. The pharmaceutical composition ofclaim 1, wherein it is orally administered either as capsules, tablets,softgels, beadlets, liquid solution, liquid suspension, liquid emulsionor as powder sachets for enhanced bioavailability of the bioactivemolecules.
 15. The pharmaceutical composition of claim 1, wherein it isadministered orally at a dose of 100 to 500 mg/dose in the form oftablets, capsules or softgel.
 16. The pharmaceutical composition ofclaim 1, wherein it is administered orally at a dose of 1000 mg to 5000mg per sachet, once or twice a day, to deliver minimum 100 to 500 mg ofbioactive molecules along with 1 to 10 g of peptides/dosage/day.
 17. Thepharmaceutical composition of claim 14, wherein the collagen bioactivemolecule complex is amorphous.
 18. A method of preparing water solublepharmaceutical composition of claim 1, comprising the steps of: a.micronisation of curcuminoids followed by emulsification byhomogenisation with an emulsifier; b. dissolving collagen peptides inwater at a concentration range of 20 to 95%; c. complex formation ofcurcuminoids with the collagen peptide matrix by sonication withemulsified curcuminoids from step (a) with the dissolved collagenpeptides from step (b) d. encapsulation of the complex from step (c) bymaking sub-micron sized micelle suspension by ultrasound mediatedhomogenisation; and e. drying the submicron sized micelles from step (d)by spray drying or freeze drying.
 19. The method of claim 18, whereinthe emulsifier is selected from the group consisting of lecithin,polysorbate, propylene glycol, sorbitol, glycerol, polyglycerol esters,Quillaja extract, and sugar esters.